PROJECT SUMMARY ? RP4 Mycobacterium tuberculosis remains one of the most devastating human infectious diseases, causing two million deaths annually and latently infecting a third of the world?s population. M. tuberculosis has recently evolved to become resistant to multiple first-line antibiotics, and as such, developing approaches to facilitate the killing of drug-resistant M. tuberculosis are needed. One such approach for which the bacteria cannot evolve resistance is to develop therapeutics that harness the host response, enhancing the body?s own systems for killing M. tuberculosis. The host process called degradative autophagy by which cells engulf and degrade intracellular bacteria has recently emerged as a possible target for host-directed therapy. In the prior CETR, together with RP1-3, we identified and characterized several molecules that can harness the autophagy pathway to restrict M. tuberculosis replication in macrophages and in vivo. We also discovered molecular mechanisms by which autophagy is initiated and bacteria targeted for degradation. Specifically, during M. tuberculosis infection, cytoplasmic DNA is detected by the proteins cGAS and STING, leading to autophagy initiation, while M. tuberculosis is targeted for degradation by the ubiquitin ligase Smurf1. In addition, we discovered that a core autophagy protein, ATG5, suppressed pathologic neutrophilic inflammation in the context of M. tuberculosis infection. Thus, in the proposed research we will (1) Test autophagy-directed compounds for activity against M. tuberculosis in preclinical models, (2) Define and target mechanisms of cGAS-STING-dependent autophagy activation during M. tuberculosis infection, (3) Define and target mechanisms by which M. tuberculosis is targeting to autophagosomes by Smurf1 and (4) Define and target pathways involved in ATG5-mediated control of neutrophilic inflammation during M. tuberculosis infection. We anticipate that these approaches will result in identification of lead compounds for future studies to establish new broad- spectrum anti-infectives.